The major goal of R01 AI42382 "Structure and Function of the HIV-1 Envelope protein" has been to uncover the mechanism of gp41 activation and HIV-1 membrane fusion. Over the past eight years, we have focused on defining the detailed structure of the fusogenic conformation of gp41, its thermodynamic and kinetic properties, its role in viral entry, and its potential as a target for specific inhibitors of HIV-1 entry. Our current studies are directed at determining the essential structural and physicochemical features of the native conformation of gp41 and its intermediates on the pathway to the fusogenic state. These experiments are designed to elucidate the precise mechanisms that regulate gp41 activation and reorganization and to develop new strategies to intervene in this process. The conserved membrane-proximal external region of the gp41 ectodomain bears the epitopes of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10, and is therefore an important target of HIV-1 vaccine design. However, all attempts to elicit comparable anti-gp41 antibodies have thus far failed. Our approach to immunogen design is guided by our recent identification and structural determination of a novel four-stranded coiled-coil domain (C43) encompassing the 2F5 epitope. This study. The overall goal of this work is to gain a detailed understanding of the structural and thermodynamic properties of the C43 coiled-coil domain, and to use this knowledge to engineer and produce stable immunogens for the induction of broadly neutralizing HIV-1 antibodies. Our central hypothesis is that presentation of the highly conserved 2F5 epitope sequence in this new structural state opens the most promising avenue for discovery and development of novel immunogens to elicit neutralizing antibodies against primary HIV-1 isolates. We propose two Specific Aims. (1) To design and develop stabilized versions of the C43 coiled-coil domain bearing the 2F5 epitope. We will generate a single- chain analog of the tetrameric coiled coil in which the four helices are connected by short peptide linkers. We will also use leucine-scanning mutagenesis to identify specific residue substitutions that favorably influence inter-helical packing interactions in the tetramer in order to stabilize an ordered helical peptide structure of the 2F5 epitope that preserves surface-exposed side chains. (2) To evaluate the immunological responses elicited by stabilized C43 coiled-coil variants in small animals. We will conduct immunogenicity studies in rabbits and guinea-pigs to determine whether the stabilized tetramer structure can elicit neutralizing antibodies. These studies will involve immunization of the animals using the stabilized C43 peptides. We will also evaluate the immunogenicity of the stable coiled-coil molecules captured onto nanometer-sized beads as particulate immunogens. The research and public health communities concur that a preventive vaccine is the obvious long-term solution to bring the global HIV-1 epidemic under control (1-8). Unfortunately, this goal has proven elusive and no such vaccine is available. Overcoming this important biomedical problem will require new and imaginative design strategies to bring us closer to the goal of a successful AIDS vaccine. [unreadable] [unreadable] [unreadable]